A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.

Leukemia is a tumor of blood-forming tissues including bone marrow and lymphatic nodes, which comprise biologically distinct subgroups. In the present study, Au NPs-PEG-Lectin was prepared as a drug targeting system for potential Thomsen-Friedenreich antigen (TF-Ag) presented on the surface of leukemic cells to induce cytotoxicity. Gold nanoparticles were prepared using citrate reduction method and conjugated with lectin via SH-PEG-COOH. The conjugate was characterized using UV/Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Zeta potential, and Scanning electron microscopy (SEM) with subsequent applications for cytotoxicity, cell cycle analysis, and apoptosis. Immunophenotypically blood samples from patients with acute lymphoblastic leukemia (ALL) were positively expressed CD45, CD95 dim expression, and low CD176 (TF-Ag) expression. Samples of acute myeloid leukemia (AML) confirmed the expression of all markers. Au NPs-PEG-Lectin conjugate showed an average size of 35.82 nm with zeta potential of -27.33 with accelerated lectin release from the conjugate at acidic pH. Au NPs-PEG-Lectin demonstrated the highest and most significant cytotoxic activity against HL-60 and K562 with IC50 of 132.5 and 314.8 μg mL-1, respectively. Flow cytometric analysis revealed induction of HL-60 cell apoptosis upon conjugate treatment in a dose-dependent pattern up to 51.03 % with no sign of necrosis with cell cycle arrest at G0/G1 phase. HL-60 cells treated with Au NPs-PEG-Lectin exhibited inter-nucleosomal DNA fragmentation. Morphologically, Phospho-Histone/BrdU dual staining indicated that Au NPs-PEG-Lectin initiated HL-60 arrest at G0/G1 phase. Taken together, molecular docking verified the possible interaction between lectin amino acids and different hydroxy groups within TF-Ag forming hydrogen bonds.

Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells mainly treated with chemotherapy for cancer remission, but this non-selective treatment also induces numerous side effects. Investigations with bioactive compounds from plant-derived foods against cancer have increased in the last years because there is an urgent need to search for new anti-leukemic agents possessing higher efficacy and selectivity for AML cells and fewer negative side effects. In this study, we analyzed the anti-leukemic activity of several phytochemicals that are representative of the major classes of compounds present in cruciferous foods (glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols, and anthocyanins) in the human acute myeloid leukemia cell line HL-60. Our results revealed that among the different Brassica-derived compounds assayed, sulforaphane (SFN) (an aliphatic isothiocyanate) showed the most potent anti-leukemic activity with an IC50 value of 6 µM in dose-response MTT assays after 48 h of treatment. On the other hand, chlorogenic acid (a hydroxycinnamic acid) and cyanidin-3-glucoside (an anthocyanin) also displayed anti-leukemic potential, with IC50 values of 7 µM and 17 µM after 48 h of incubation, respectively. Importantly, these compounds did not show significant cell toxicity in macrophages-like differentiated cells at 10 and 25 µM, indicating that their cytotoxic effects were specific to AML cancer cells. Finally, we found that these three compounds were able to induce the NRF2/KEAP1 signaling pathway in a dose-dependent manner, highlighting SFN as the most potent NRF2 activator. Overall, the present evidence shed light on the potential for using foods and ingredients rich in anticancer bioactive phytochemicals from Brassica spp.

Acute myeloid leukemia (AML)-the most frequent form of adult blood cancer-is characterized by heterogeneous mechanisms and disease progression. Developing an effective therapeutic strategy that targets metabolic homeostasis and energy production in immature leukemic cells (blasts) is essential for overcoming relapse and improving the prognosis of AML patients with different subtypes. With respect to metabolic regulation, fructose-1,6-bisphosphatase 1 (FBP1) is a gluconeogenic enzyme that is vital to carbohydrate metabolism, since gluconeogenesis is the central pathway for the production of important metabolites and energy necessary to maintain normal cellular activities. Beyond its catalytic activity, FBP1 inhibits aerobic glycolysis-known as the "Warburg effect"-in cancer cells. Importantly, while downregulation of FBP1 is associated with carcinogenesis in major human organs, restoration of FBP1 in cancer cells promotes apoptosis and prevents disease progression in solid tumors. Recently, our large-scale sequencing analyses revealed FBP1 as a novel inducible therapeutic target among 17,757 vitamin-D-responsive genes in MV4-11 or MOLM-14 blasts in vitro, both of which were derived from AML patients with FLT3 mutations. To investigate FBP1's anti-leukemic function in this study, we generated a new AML cell line through lentiviral overexpression of an FBP1 transgene in vitro (named FBP1-MV4-11). Results showed that FBP1-MV4-11 blasts are more prone to apoptosis than MV4-11 blasts. Mechanistically, FBP1-MV4-11 blasts have significantly increased gene and protein expression of P53, as confirmed by the P53 promoter assay in vitro. However, enhanced cell death and reduced proliferation of FBP1-MV4-11 blasts could be reversed by supplementation with post-glycolytic metabolites in vitro. Additionally, FBP1-MV4-11 blasts were found to have impaired mitochondrial homeostasis through reduced cytochrome c oxidase subunit 2 (COX2 or MT-CO2) and upregulated PTEN-induced kinase (PINK1) expressions. In summary, this is the first in vitro evidence that FBP1-altered carbohydrate metabolism and FBP1-activated P53 can initiate leukemic death by activating mitochondrial reprogramming in AML blasts, supporting the clinical potential of FBP1-based therapies for AML-like cancers.

Acute myeloid leukemia (AML) is one of the most common malignancies of the hematopoietic progenitor cell in adults. Quercetin has gained recognition over the years because of its anti-cancer effect with minimal toxicity. Herein, we aim to investigate the anti-leukemia mechanism of quercetin and to decipher the signaling pathway of quercetin in HL-60 leukemic cells. We observed that quercetin induces apoptosis and autophagic cell death, in which both pathways play an important role in suppressing the viability of leukemia cells. Phosphorylated AMPK (p-AMPK) protein expressions are lower in primary AML cells, HL-60 cells, KG-1 and THP-1 cells than in peripheral blood monocular cells. After quercetin treatment, the expression of p-AMPK is increased while the expression of p-mTOR is decreased in a dose-dependent manner. Mechanistically, compound C, an AMPK phosphorylation inhibitor, upregulates the phosphorylation of mTOR and inhibits autophagy and apoptosis in quercetin-induced HL-60 cells, while silencing of CaMKKβ inhibits the quercetin-induced phosphorylation of AMPK, resulting in increased mTOR phosphorylation. Furthermore, silencing of CaMKKβ inhibits the autophagy in HL-60 cells. Taken together, our data delineate that quercetin plays its anti-leukemia role by inhibiting cell viability and inducing apoptosis and autophagy in leukemia cells. Quercetin inhibits the phosphorylation of mTOR by regulating the activity of AMPK, thus playing a role in the regulation of autophagy and apoptosis. CaMKKβ is a potential upstream molecule for AMPK/mTOR signaling pathway, through which quercetin induces autophagy in HL-60 cells.

Acute myelocytic leukemia (AML) is a malignancy of the stem cell precursors of the myeloid lineage. CD4+ and CD8+ T cells play pivotal roles in influencing AML progression but are functionally suppressed in the bone marrow microenvironment. We aimed to find hub genes related to T cell exhaustion and suppression, thereby providing evidence for immunotherapy. In this study, gene transcriptome expression data from TCGA and TARGET databases were utilized to find key genes. Firstly, CIBERSORT immune cell infiltration algorithm and WGCNA method were used to identify CD4+ and CD8+ T cells-related genes. Univariate and multivariate cox regression analyses were then introduced to construct the overall survival prognosis model and included hub genes. The ESTIMATE and ssGSEA scoring methods were used to analyze the correlation between the hub genes and immune activity. Single-cell transcriptome analysis was applied to detect the immune cells expressing hub genes, hence, to detect exact mechanisms. Consequently, FLT3LG and IFITM3P6 were determined to be positively correlated with patients' overall survival and microenvironment immune activity. Further study suggested FLT3-FLT3LG and IFITM3P6-miR-6748-3p-CBX7 signaling axes were involved in CD4+ and CD8+ T cells activation. This may be one of the mechanisms of T cells suppression in AML.

Acute myeloid leukemia (AML) is prone to relapse. Targeted therapy with a specific inhibitor of the anti-apoptotic protein Bcl-2 ABT-199 is an effective method for relapsed and refractory patients, but drug resistance is likely, which is primarily related to high Mcl-1 and S100A8 expression. All-trans retinoic acid (ATRA) can inhibit Bcl-2 and Mcl-1 expression. The study purpose was to determine whether ATRA can enhance the antileukemia effect of ABT-199 on AML cells. Our data showed that ATRA combined with ABT-199 exerts a synergistic antileukemic effect by inducing apoptosis and cell cycle arrest in AML. In vivo, combination therapy prolonged the survival of AML xenograft mice. The possible mechanism involves promoting apoptosis through downregulation of S100A8 expression by inhibiting the PI3K/AKT signaling pathway. This study provides a potential treatment strategy and theoretical support for overcoming the clinical ABT-199 resistance problem in AML patients.

Minimal residual disease (MRD) detection is a strong predictor for survival and relapse in acute myeloid leukemia (AML). MRD can be either determined by molecular assessment strategies or via multiparameter flow cytometry. The degree of bone marrow (BM) dilution with peripheral blood (PB) increases with aspiration volume causing consecutive underestimation of the residual AML blast amount. In order to prevent false-negative MRD results, we developed Cinderella, a simple automated method for one-tube simultaneous measurement of hemodilution in BM samples and MRD level. The explainable artificial intelligence (XAI) Cinderella was trained and validated with the digital raw data of a flow cytometric "8-color" AML-MRD antibody panel in 126 BM and 23 PB samples from 35 patients. Cinderella predicted PB dilution with high accordance compared to the results of the Holdrinet formula (Pearson's correlation coefficient r = 0.94, R2 = 0.89, p < 0.001). Unlike conventional neuronal networks Cinderella calculated the distributions of 12 different cell populations that were assigned to true hematopoietic counterparts as a human in the loop (HIL) approach. Besides characteristic BM cells such as myelocytes and myeloid progenitor cells the XAI identified discriminating populations, which were not specific for BM or PB (e.g., T cell/NK cell subpopulations and CD45 negative cells) and considered their frequency differences. Thus, Cinderella represents a HIL-XAI algorithm capable to calculate the degree of hemodilution in BM samples with an AML MRD immunophenotype panel. It is explicable, transparent, and paves a simple way to prevent false negative MRD reports.

The four and a half LIM domains 1 (FHL1) is considered to play important roles in tumors. This study aims to investigate the role and precise mechanisms of FHL1 in acute myeloid leukemia (AML). Here, we found that FHL1 was highly expressed in AML. CCK8, flow cytometry, and Western blot analysis of cell cycle-related proteins showed that overexpression of FHL1 promoted proliferation and accelerated cell cycle progression in HL-60 cells. Conversely, knockdown of FHL1 inhibited the proliferation and induced cell cycle arrest in KG-1 cells. Furthermore, knockdown of FHL1 promoted cell differentiation, while overexpression of FHL1 restrained all-trans retinoic acid induced cell differentiation in HL-60 cells, revealed by Wright-Giemsa staining and cell surface antigen analysis. Moreover, in vivo experiments revealed that depletion of FHL1 inhibited tumor growth and led to increased levels of CD11b and CD14. Here, we first identify an unexpected and important role of FHL1 that contributes to the AML progression, indicating that FHL1 may be a potential therapeutic target for AML.

Background: Acute myelocytic leukemia (AML) is one of the hematopoietic cancers with an unfavorable prognosis. However, the prognostic value of N 6-methyladenosine-associated long non-coding RNAs (lncRNAs) in AML remains elusive. Materials and Methods: The transcriptomic data of m6A-related lncRNAs were collected from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. AML samples were classified into various subgroups according to the expression of m6A-related lncRNAs. The differences in terms of biological function, tumor immune microenvironment, copy number variation (CNV), and drug sensitivity in AML between distinct subgroups were investigated. Moreover, an m6A-related lncRNA prognostic model was established to evaluate the prognosis of AML patients. Results: Nine prognosis-related m6A-associated lncRNAs were selected to construct a prognosis model. The accuracy of the model was further determined by the Kaplan-Meier analysis and time-dependent receiver operating characteristic (ROC) curve. Then, AML samples were classified into high- and low-risk groups according to the median value of risk scores. Gene set enrichment analysis (GSEA) demonstrated that samples with higher risks were featured with aberrant immune-related biological processes and signaling pathways. Notably, the high-risk group was significantly correlated with an increased ImmuneScore and StromalScore, and distinct immune cell infiltration. In addition, we discovered that the high-risk group harbored higher IC50 values of multiple chemotherapeutics and small-molecule anticancer drugs, especially TW.37 and MG.132. In addition, a nomogram was depicted to assess the overall survival (OS) of AML patients. The model based on the median value of risk scores revealed reliable accuracy in predicting the prognosis and survival status. Conclusion: The present research has originated a prognostic risk model for AML according to the expression of prognostic m6A-related lncRNAs. Notably, the signature might also serve as a novel biomarker that could guide clinical applications, for example, selecting AML patients who could benefit from immunotherapy.

Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by variations in cytogenetics and molecular abnormalities, which result in variable response to therapy. Receptor-interacting serine/threonine kinase 1 (RIP1)-mediated necroptosis has been reported to have a potential role in the treatment of AML. We obtained Skp2 and RIP1 are significantly overexpressed in AML samples using original published data, and identified that Skp2-depletion in AML cells significantly suppressed RIP1. Functional analysis showed that the inhibition of RIP1 caused by necrostatin-1 (Nec-1) inhibited the proliferation, simultaneously facilitate both the apoptosis and differentiation of AML cells. Mechanistical analysis elucidated that knockdown of Skp2 suppresses RIP1 by transcriptional regulation but not by proteasome degradation. Additionally, Skp2 regulated the function of RIP1 by decreasing K63-linked ubiquitin interaction with RIP1. Moreover, the suppression of Akt/GSK3β was observed in Skp2 knockdown stable NB4 cells. Also, GSK3β inactivation via small-molecule inhibitor treatment remarkably decreased RIP1 level. RIP1 regulates differentiation by interacting with RARα, increasing RA signaling targets gene C/EBPα and C/EBPβ. In conclusion, our study provides a novel insight into the mechanism of tumorigenesis and the development of AML, for which the Skp2-Akt/GSK3β-RIP1 pathway can be developed as a promising therapeutic target.

This study mainly used The Cancer Genome Atlas (TCGA) RNA sequencing dataset to screen prognostic snoRNAs of acute myeloid leukemia (AML), and used for the construction of prognostic snoRNAs signature for AML. A total of 130 AML patients with RNA sequencing dataset were used for prognostic snoRNAs screenning. SnoRNAs co-expressed genes and differentially expressed genes (DEGs) were used for functional annotation, as well as gene set enrichment analysis (GSEA). Connectivity Map (CMap) also used for potential targeted drugs screening. Through genome-wide screening, we identified 30 snoRNAs that were significantly associated with the prognosis of AML. Then we used the step function to screen a prognostic signature composed of 14 snoRNAs (SNORD72, SNORD38, U3, SNORA73B, SNORD79, SNORA73, SNORD12B, SNORA74, SNORD116-12, SNORA65, SNORA14, snoU13, SNORA75, SNORA31), which can significantly divide AML patients into high- and low-risk groups. Through GSEA, snoRNAs co-expressed genes and DEGs functional enrichment analysis, we screened a large number of potential functional mechanisms of this prognostic signature in AML, such as phosphatidylinositol 3-kinase-Akt, Wnt, epithelial to mesenchymal transition, T cell receptors, NF-kappa B, mTOR and other classic cancer-related signaling pathways. In the subsequent targeted drug screening using CMap, we also identified six drugs that can be used for AML targeted therapy, they were alimemazine, MG-262, fluoxetine, quipazine, naltrexone and oxybenzone. In conclusion, our current study was constructed an AML prognostic signature based on the 14 prognostic snoRNAs, which may serve as a novel prognostic biomarker for AML.

C/EBPβ has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPβ an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPβ. The synthetic compound inhibits C/EBPβ by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPβ or MYB and gene expression profiling validate C/EBPβ as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPβ and identifies the cysteine residues in the transactivation domain of C/EBPβ as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPβ inhibitors before being used as a therapeutic agent.

Over half of older patients with acute myeloid leukemia (AML) do not respond to cytotoxic chemotherapy, and most responders relapse because of drug resistance. Cytarabine is the main drug used for the treatment of AML. Intensive treatment with high-dose cytarabine can increase the overall survival rate and reduce the relapse rate, but it also increases the likelihood of drug-related side effects. To optimize cytarabine treatment, understanding the mechanism underlying cytarabine resistance in leukemia is necessary. In this study, the gene expression profiles of parental HL60 cells and cytarabine-resistant HL60 (R-HL60) cells were compared through gene expression arrays. Then, the differential gene expression between parental HL60 and R-HL60 cells was measured using KEGG software. The expression of numerous genes associated with the nuclear factor κB (NF-κB) signaling pathway changed during the development of cytarabine resistance. Proteasome inhibitors inhibited the activity of non-canonical NF-κB signaling pathway and induced the apoptosis of R-HL60 cells. The study results support the application and possible mechanism of proteasome inhibitors in patients with relapsed or refractory leukemia.

Acute myeloid leukemia (AML) has become a worldwide malignant cancer. We intended to investigate the critical roles and mechanism underlying homo sapiens circular RNA 0003602 (hsa_circ_0003602) in AML progression, especially in tumor cell proliferation, migration, invasion, and apoptosis. Real-time PCR was applied to identify the differential expression of hsa_circ_0003602 and miR-502-5p in AML bone marrow tissues and cell lines. In addition, western blot analysis was employed to determine the levels insulin-like growth factor 1 receptor (IGF1R) protein. The biological behaviors were assessed by CCK-8 cell viability assay, flow cytometry assay for apoptosis detection, and Transwell migration and invasion assay. The relationships between target miRNA and downstream mRNA were investigated by bioinformatics, luciferase reporter assay, and biotin-labeled RNA pull-down assay. Hsa_circ_0003602 was upregulated and predicted poor survival in AML. Knockdown of hsa_circ_0003602 in AML cell lines induced the inhibition of proliferation, migration, and invasion and caused apoptosis. Hsa_circ_0003602 sequestered miR-502-5p by functioning as a competitive endogenous RNA (ceRNA), thereby regulating IGF1R expression. Hsa_circ_0003602 acted as a tumor promoter in AML via miR-502-5p/IGF1R axis. Our study provides evidence that hsa_circ_0003602, miR-502-5p, and IGF1R might form a regulatory axis to affect the carcinogenicity of AML cells and provide potential targets for the treatment of AML.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal hematologic malignancy for which novel therapeutic targets and strategies are required. Emerging evidence suggests that WTIP is a candidate tumor suppressor. However, the molecular mechanisms of WTIP in leukemogenesis have not been explored. Here, we report that WTIP expression is significantly reduced both in AML cell lines and clinical specimens compared with normal controls, and low levels of WTIP correlate with decreased overall survival in AML patients. Overexpression of WTIP inhibits cell proliferation and induces apoptosis both in vitro and in vivo. Mechanistic studies reveal that the apoptotic function of WTIP is mediated by upregulation and nuclear translocation of FOXO3a, a member of Forkhead box O (FOXO) transcription factors involved in tumor suppression. We further demonstrate that WTIP interacts with FOXO3a and transcriptionally activates FOXO3a. Upon transcriptional activation of FOXO3a, its downstream target PUMA is increased, leading to activation of the intrinsic apoptotic pathway. Collectively, our results suggest that WTIP is a tumor suppressor and a potential target for therapeutic intervention in AML.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.